AF594 Picolyl Azide

Guide (The following is our recommended protocol. This protocol is for guidance only and should be modified according to your specific needs).

1. Stock Solution Preparation
1.1 Solvent: DMSO.
1.2 Concentration recommendation: 2 mM[1], 100 μM[2].

2. Working Solution Preparation
2.1 Diluent: Click buffer containing 100 mM K₂HPO₄, 150 µM CuSO₄, 300 µM BTTAA, 2.5 mM sodium ascorbate; 1× Click-iT cocktail.
2.2 Working concentration: 10 µM; used as part of 1× Click-iT cocktail.
2.3 Note: Adjust working solution concentration as needed; prepare fresh before use.

3. Staining Procedure
3.1 For adherent human cell lines (CCD841CoN, HT29, HCT116, HEK293, HeLa)[1]:
3.1.1 Sample preparation: Rinse cell monolayers with ice-cold PBS, gently scrape cells into DPBS^−/−, then centrifuge at 600 g (CCD841CoN) or 400 g (other cell lines) for 5 min at 4°C.
3.1.2 Incubation conditions: Incubate with 10 µM AF594 Picolyl Azide working solution for 45 min at room temperature, in the dark.
3.1.3 Washing steps: Wash three times with 1 mL DPBS, centrifuging at 600 g for 5 min at 4°C after each wash.
3.2 For suspension bacterial cells (Bacillus subtilis)[2]:
3.2.1 Sample preparation: Harvest by centrifugation at 15,000g for 3 min after fixation; centrifuge at 6000g for 5 min for initial fixation step.
3.2.2 Incubation conditions: Incubate with 1× Click-iT cocktail containing AF594 Picolyl Azide for 20 min in the dark.
3.2.3 Washing steps: Wash once using Click-iT rinse buffer.

4. Detection & Analysis
4.1 Instrument type: Flow cytometer, fluorescence microscope.
4.1.1 Detection channel: FL2 channel (for flow cytometer).
4.2 Ex/Em wavelengths: Compatible with Alexa Fluor 594 (red fluorescence).
4.3 Result analysis:
4.3.1 Fluorescence intensity quantification: Measure mean fluorescence intensity (MFI) to quantify cell surface labeling efficiency (higher MFI indicates more efficient metabolic incorporation of alkyne-modified monosaccharide reporters); higher fluorescence intensity indicates higher protein synthesis rate (higher OPP incorporation).
4.3.2 Fluorescence localization: Localized to cell surface sialylated glycoconjugates; cytoplasmic (associated with nascent polypeptides).
4.3.3 Fluorescence output: Red fluorescence detected via the FL2 channel (flow cytometer); red fluorescence observed (fluorescence microscope).